Treating NIDDM with RXR agonists

ABSTRACT

This invention relates to methods and compositions for the treatment of non-insulin-dependent diabetes mellitus using an RXR agonist alone or in combination with a PPARγ agonist such as a thiazolidinedione compound.

RELATED APPLICATIONS

This is a division of application Ser. No. 08/710,309 filed Sep. 17,1996, now abandoned, which is converted from provisional applicationSer. No. 60/021,839--filed Jul. 10, 1996; Ser. No. 60/018,318--filed May24, 1996; Ser. No. 60/009,884--filed Jan. 11, 1996; Ser. No.60/004,897--filed Oct. 6, 1995; and 60/003,869--filed Sep. 18, 1995; allof which are incorporated by reference herein in their entirety,including any drawings and figures.

FIELD OF THE INVENTION

This invention relates to methods and pharmaceutical compounds fortreating diabetes and related symptoms.

BACKGROUND OF THE INVENTION

Non-insulin-dependent diabetes mellitus (NIDDM, type II diabetes) ischaracterized by abnormalities in insulin secretion and insulin action.NIDDM constitutes 90-95% of the approximately 6 million diagnoseddiabetics in the United States. NIDDM is characterized by hyperglycemia,the result of insulin resistance in peripheral tissues (skeletal muscleand adipose tissue), where insulin-stimulated uptake/utilization ofglucose is blunted, and in liver, where insulin suppression of glucoseoutput is insufficient. These impairments in insulin action play animportant role in the development of elevated fasting blood glucose andglucose intolerance.

Diet and exercise are first-line therapy for NIDDM patients. NIDDMpatients also take oral hypoglycemic drugs to control blood glucoselevels. The most widely used hypoglycemic agents are variousformulations of insulin and sulfonylureas. A major drawback with thesetherapies is the occurrence of potentially life-threatening hypoglycemiadue to hyperinsulinemia.

The hyperinsulinemia that can occur with these therapies is alsoassociated with an elevated risk of cardiovascular disease, a majorkiller of diabetics. Therefore, a need exists for antidiabetic drugswhich do not increase circulating insulin concentrations.

A new class of compounds, thiazolidinediones, have been documented toeffect antihyperglycemic activity by is increasing insulin action ratherthan by promoting insulin secretion. Thiazolidinediones ameliorateinsulin resistance and normalize plasma glucose and insulin (whereelevated) without causing a hypoglycemic state, even at very high doses.The thiazolidinedione insulin sensitizers, e.g., ciglitazone,englitazone, pioglitazone, BRL 49653(5-[[4-[2-(methyl-2-pyridinylamino)ethoxy]phenyl]methyl]-2,4-thiazolidinedione),and troglitazone, enhance insulin-mediated suppression of hepaticglucose output and insulin-stimulated glucose uptake and utilization byadipose tissue. Thiazolidinediones also change glucose transporter (e.g.Glut 4) expression to contribute to increased insulin responsiveness.

SUMMARY OF THE INVENTION

Applicant has found that RXR agonists mimic or enhance the antidiabeticeffects of thiazolidinedione compounds. RXR agonists activate thetranscriptional activity of RXR/PPARγ heterodimers, increase insulinstimulated glucose uptake, lower the level of triglyceride, suppress thelevel of insulin, and increase the level of HDL cholesterol. Two RXRagonists have been shown to lower glucose, triglycerides and insulinlevels in two established animal models of NIDDM, i.e. the ob/ob anddb/db mice. Therefore, RXR agonists may be used as insulin sensitizersor insulin mimetics in the treatment of NIDDM and related symptoms.

In addition, the combination of an RXR agonist and a PPARγ agonist, suchas a thiazolidinedione, achieves synergistic activation of the RXR/PPARγheterodimers so as to enhance adipogenic and antidiabetic effects ofPPARγ. In db/db mice, the combination of an RXR agonist and a PPARγagonist was shown to lower the level of glucose more than individualcompounds did.

Therefore, the present invention relates to methods and compositions fortreating a host having NIDDM or insulin resistant diabetes byadministering to the host a composition containing a pharmaceuticallyeffective amount of an activator of the RXR/PPARγ heterodimer,including, but not limited to, an RXR agonist. The host may be a humanpatient or an animal model of human NIDDM. The compositions of thisinvention are adapted to cure, improve or prevent one or more symptomsof NIDDM in the host. A preferred drug is highly potent and selectivewith low toxicity. In this regard, those skilled in the art willrecognize NIDDM as an example of a metabolic disease that can be treatedwith the RXR agonist-containing compounds and compositions of thepresent invention. Other examples of metabolic diseases treatable withthe compounds and compositions of the present invention include, but arenot limited to, obesity and thyroid hormone abnormalties.

By "pharmaceutically effective amount" is meant an amount of apharmaceutical compound or composition having a therapeutically relevanteffect on NIDDM. A therapeutically relevant effect relieves to someextent one or more symptoms of NIDDM in a patient or returns to normaleither partially or completely one or more physiological or biochemicalparameters associated with or causative of NIDDM, e.g. increasing thesensitivity of cellular response to circulating insulin, curing,reducing, or preventing one or more clinical symptoms of NIDDM,including, but not limited to, hyperglycemia, hyperinsulinemia andhypertriglyceridemia. In a preferred embodiment, a pharmaceuticallyeffective amount of a compound or composition means an amount thatincreases the uptake of glucose by adipose tissue or muscle tissue. Inanother preferred embodiment, a pharmaceutically effective amount of acompound or composition means an amount that increases the uptake oftriglyceride by adipose tissue.

By "activator of the RXR/PPRγ heterodimer" is meant a compound orcomposition which when combined with the RXR/PPARγ heterodimer increasesthe transcriptional regulation activity of the heterodimer, as measuredby an assay known to one skilled in the art, including, but not limitedto, the "co-transfection" or "cis-trans" assays described or disclosedin U.S. Pat. Nos. 4,981,784, 5,071,773, 5,298,429, 5,506,102,WO89/05355, WO91/06677, WO92/05447, WO93/11235, WO95/18380,PCT/US93/04399, PCT/US94/03795 and CA 2,034,220, which are incorporatedby reference herein. It includes, but is not limited to, compounds thatbind RXR, PPARγ, or both.

By "RXR agonist" is meant a compound or composition which when combinedwith RXR homodimers or heterodimers increases the transcriptionalregulation activity of RXR, as measured by an assay known to one skilledin the art, including, but not limited to, the "co-transfection" or"cis-trans" assays described or disclosed in U.S. Pat. Nos. 4,981,784,5,071,773, 5,298,429, 5,506,102, WO89/05355, WO91/06677, WO92/05447,WO93/11235, WO95/18380, PCT/US93/04399, PCT/US94/03795 and CA 2,034,220,which are incorporated by reference herein. It includes, but is notlimited to, compounds that preferentially activate RXR over RAR (i.e.RXR specific agonists), and compounds that activate both RXR and RAR(i.e. pan agonists). It also includes compounds that activate RXR in acertain cellular context but not others (i.e. partial agonists).Compounds disclosed or described in the following articles, patents andpatent applications which have RXR agonist activity are incorporated byreference herein: U.S. Pat. Nos. 5,399,586 and 5,466,861, WO96/05165,PCT/US95/16842, PCT/US95/16695, PCT/US93/10094, WO94/15901,PCT/US92/11214, WO93/11755, PCT/US93/10166, PCT/US93/10204, WO94/15902,PCT/US93/03944, WO93/21146, provisional applications Ser. No. 60,004,897and 60,009,884, Boehm, et al. J. Med. Chem. 38(16):3146-3155, 1994,Boehm, et al. J. Med. Chem. 37(18):2930-2941, 1994, Artras et al., J.Biol. Chem. 266:1157-1161 (1991), Salazar-Olivo et al., Biochem.Biophys. Res. Commun. 204:157-263 (1994) and Safanova, Mol. Cell.Endocrin. 104:201-211 (1994). RXR specific agonists include, but are notlimited to, LG 100268 (i.e.2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-cyclopropyl]-pyridine-5-carboxylicacid) and LGD 1069 (i.e.4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-2-carbonyl]-benzoicacid), and analogs, derivatives and pharmaceutically acceptable saltsthereof. The structures and syntheses of LG 100268 and LGD 1069 aredisclosed in Boehm, et al. J. Med. Chem. 38(16):3146-3155, 1994,incorporated by reference herein. Pan agonists include, but are notlimited to, ALRT 1057 (i.e. 9-cis retinoic acid), and analogs,derivatives and pharmaceutically acceptable salts thereof.

In a preferred embodiment, the pharmaceutical composition also containsa pharmaceutically effective amount of a PPARγ agonist. Alternatively, asecond composition containing a pharmaceutically effective amount of aPPARγ agonist is administered to the host separately. In a furtherpreferred embodiment, a compound having agonist activity for both RXRand PPARγ is used.

By "PPARγ agonist" is meant a compound or composition which whencombined with PPARγ increases a reaction typical for the receptor, e.g.,transcriptional regulation activity, as measured by an assay known toone skilled in the art, including, but not limited to, the"co-transfection" or "cis-trans" assays described or disclosed in U.S.Pat. Nos. 4,981,784 and 5,071,773 and Lehmann, et al., J. Biol. Chem.270:12953-12956 (1995), which are incorporated by reference herein. Apreferred PPARγ agonist is a thiazolidinedione compound, including, butnot limited to, BRL 49653, troglitazone, pioglitazone, ciglitazone,WAY-120,744, englitazone, AD 5075, darglitazone, and analogs,derivatives and pharmaceutically acceptable salts thereof. Compoundsdisclosed in Tontonez et al., Genes & Develop. 8:1224-1234 (1994),Tontonez et al., Cell 79:1147-1156 (1994), Lehmann et al., J. Biol.Chem. 270(22):1-4, 1995, Amri et al., J. Lipid Res. 32:1449-1456 (1991),Amri et al., J. Lipid Res. 32:1457-1463, (1991) and Grimaldi et al.,Proc. Natl. Acad. Sci, USA 89:10930-10934 (1992) are incorporated byreference herein.

In a further preferred embodiment, the pharmaceutical composition alsocontains a pharmaceutically effective amount of insulin, insulinderivative, insulin secretagogue, insulin sensitizer, or insulinmimetic. Alternatively, a composition containing a pharmaceuticallyeffective amount of insulin, insulin derivative, insulin secretagogue,insulin sensitizer, or insulin mimetic is administered to the hostseparately.

A composition containing a pharmaceutically effective amount of anactive ingredient may be administered orally or systemically to a host.In a preferred embodiment, it is administered orally.

In another aspect, this invention features a pharmaceutical compositionfor treating NIDDM containing a pharmaceutically effective amount of anRXR agonist; and a pharmaceutically acceptable carrier adapted for ahost having NIDDM. In a preferred embodiment, the pharmaceuticalcomposition also includes a pharmaceutically effective amount ofinsulin, insulin derivative, insulin secretagogue, insulin sensitizer,insulin mimetic or PPARγ agonist.

In a preferred embodiment, the composition is held within a containerwhich includes a label stating to the effect that the composition isapproved by the FDA in the United States (or an equivalent regulatoryagency in a foreign country) for treating NIDDM or for treatinghyperglycemia, hyperinsulinemia or hypertriglyceridemia. Such acontainer provides a therapeutically effective amount of the activeingredient to be administered to a host.

In another aspect, this invention features methods for screening forcandidate compounds useful for treating NIDDM. These methods selectcompounds or compositions which when combined with the RXR/PPARγheterodimer increase the transcriptional regulation activity of theheterodimer, as measured by an assay known to one skilled in the art,including, but not limited to, the "co-transfection" or "cis-trans"assays described or disclosed in U.S. Pat. Nos. 4,981,784, 5,071,773,5,298,429, 5,506,102, WO89/05355, WO91/06677, WO92/05447, WO93/11235,WO95/18380, PCT/US93/04399, PCT/US94/03795 and CA 2,034,220, which areincorporated by reference herein. In one example, a candidate compoundsuch as a potential RXR agonist is administered to an adipocyte or apreadipocyte. The level of lipid in the cell is measured, and anincreased accumulation of lipid after the treatment with the candidatecompound indicates that the candidate compound is useful for treatingNIDDM. In preferred embodiments, the level of lipid is measured by oilred O staining or detecting the level of triglyceride in the cell.

In another example, a candidate compound such as a potential RXR agonistis administered to an adipocyte or a preadipocyte and the transcriptionlevel of a adipocyte specific gene (e.g. lipoprotein lipase gene orPPARγ gene) is measured. An increased transcription of the adipocytespecific gene after the treatment with the candidate compound indicatesthat the candidate compound is useful for treating NIDDM.

In yet another example, a candidate compound such as a potential RXRagonist is administered to an adipocyte or a preadipocyte and the levelof glucose uptake is measured. An increased glucose uptake after thetreatment with the candidate compound indicates that the candidatecompound is useful for treating NIDDM. Alternatively, both the candidatecompound and insulin are administered to the cell and the level ofglucose uptake is compared to that in the same cell treated with insulinalone. A higher level of glucose uptake in the cell treated by thecandidate compound and insulin indicates that the candidate compound isan insulin sensitizer and useful for treating NIDDM.

Other features and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a graph showing the extent of adipocyte differentiation in3T3-LI cells as measured by the levels of triglyceride in 3T3-LIpreadipocytes treated with various combinations of an RXR agonist (LG100268), a PPARγ agonist (thiazolidinedione compound BRL 49653), andinsulin. The retinoid and BRL 49653 were used at 1 μM.

FIG. 1b is a graph showing the extent of adipocyte differentiation in3T3-LI cells as measured by the level of triglyceride in 3T3-LIpreadipocytes treated with various combinations of an RXR agonist (LGD1069), a PPARγ agonist (thiazolidinedione compound BRL 49653), andinsulin. The retinoid and BRL 49653 were used at 1 μM.

FIG. 2a is a graph showing the level of LPL mRNA in 3T3-LI cells treatedwith various combinations of an RXR agonist (LG 100268), a PPARγ agonist(thiazolidinedione compound BRL 49653), and insulin. The retinoid andBRL 49653 were used at 1 μM.

FIG. 2b is a graph showing the level of PPARγ mRNA in 3T3-LI cellstreated with various combinations of an RXR agonist (LG 100268), a PPARγagonist (thiazolidinedione compound BRL 49653), and insulin. Theretinoid and BRL 49653 were used at 1 μM.

FIG. 3 is a graph showing the level of glucose in db/db mice treatedwith LG 100268, BRL 49653, and a combination of LG 100268 and BRL 49653,respectively.

FIG. 4 is a graph showing the level of triglycerides in db/db micetreated with LG 100268, BRL 49653, and a combination of LG 100268 andBRL 49653, respectively.

FIG. 5 is a graph showing the level of HDL cholesterol in db/db micetreated with LG 100268, BRL 49653, and a combination of LG 100268 andBRL 49653, respectively.

FIG. 6 is a graph showing the level of triglycerides in ob/ob micetreated with LGD 1069, LG 100268, and BRL 49653, respectively.

FIG. 7 is a graph showing the level of glucose in ob/ob mice treatedwith LGD 1069, LG 100268, and BRL 49653, respectively.

FIG. 8 is a graph showing the level of insulin in ob/ob mice treatedwith LGD 1069, LG 100268, and BRL 49653, respectively.

DETAILED DESCRIPTION OF THE INVENTION

TZD Achieves Antidiabetic and Adipogenic Effects through PPARγ

Thiazolidinediones are insulin sensitizers that significantly reduceglucose and lipid levels in animal models of NIDDM and obesity (Kees, etal., J. Medicinal Chem. 38(4):617-628, 1995; Willson, et al., J.Medicinal Chem. 39(3):665-668, 1996; Young, et al. Diabetes44:1087-1092, 1995). Thiazolidinediones improve glucose utilizationwithout stimulating insulin release.

For example, repeated administration of BRL 49653 to obese mice improvesglycemic control by increasing insulin responsiveness of target tissues.BRL 49653 potentiates insulin-stimulated glucose transport in adipocytesfrom insulin-resistant obese mice, both by increasing insulin receptornumber and by facilitating translocation of GLUT4, from an expandedintracellular pool, to the cell surface.

Thiazolidinediones are also selective PPARγ agonists (Lehmann, et al. J.Biol. Chem. 270(22):1-4, 1995). Comparison of the EC₅₀ for activation ofPPARγ with the minimum effective dose (MED) for anti-hyperglycemicactivity revealed a significant correlation. The correlation between invitro PPARγ activity and in vivo antihyperglycemic activity ofthiazolidinediones implicates PPARγ as the molecular target for theantidiabetic effects of thiazolidinediones.

PPARγ is a member of the nuclear receptor superfamily ofligand-activated transcription factors. It is expressed in anadipose-specific manner and its expression is induced early during thedifferentiation of several preadipocyte cell lines. Forced expression ofPPARγ in fibroblasts resulted in adipocyte differentiation.

In addition to insulin sensitizing activity, thiazolidinediones havemarked adipogenic effects on preadipocyte and mesenchymal stem cells(Tontonoz, et al., Cell 79:1147-1156, 1994). Treatment of C3H/10T1/2cells with BRL 49653 resulted in efficient adipocyte differentiation,showing that ligand-mediated activation of PPARγ is sufficient toinitiate the adipogenic signaling cascade in a mesenchymal stem cellline. PPARγ is the molecular target for the adipogenic effects ofthiazolidinediones.

Adipogenesis plays a role in the development of NIDDM, which ischaracterized by not only unbalanced glucose homeostasis, but alsoelevated levels of circulating lipids. Increases in lipid levels havebeen shown to interfere with glucose disposal.

Adipocytes are highly specialized cells that play a critical role inlipid metabolism and energy homeostasis. Their primary role is to storetriglycerides in times of caloric excess and to mobilize this reserveduring periods of nutritional deprivation.

Adipocyte differentiation is characterized by a coordinate increase inadipocyte-specific gene expression. PPARγ is specifically expressed inadipocytes. Its expression is induced early during the course ofdifferentiation of several preadipocyte cell lines. Forced expression ofPPARγ in fibroblasts resulted in adipocyte differentiation.

Synergistic Effects of RXR and PPARγ on Adipogenesis

Expression of PPARγ is induced early during the differentiation ofcultured adipocyte cell lines and is expressed at very high levelsspecifically in adipose tissue. PPARγ regulates adipogenesis bymodulating the transcription of other adipocyte-specific genes, e.g.adipocyte P2 gene (aP2 gene). aP2 gene encodes an intracellularlipid-binding protein and is expressed exclusively in adipose cells.

A 518-bp DNA fragment from the 5'-flanking region of the aP2 gene hasbeen identified as an enhancer that directs high-leveladipocyte-specific gene expression in both cultured cells and transgenicmice. A pair of elements in the aP2 enhancer, ARE6 and ARE7, bind anuclear factor termed ARF6 that is detected only in nuclear extractsderived from adipocytes. The ARF6-binding sites are both necessary andsufficient for adipocyte-specific expression, suggesting that thetrans-acting factor ARF6 functions as a differentiation-dependent andtissue-specific switch for the aP2 enhancer (Tontonoz, et al. Genes &Development, 8:1224-1234, 1994).

The ARF6 recognition sequence resembles a type of nuclear hormonereceptor-binding site known as DR-1 (direct repeat with 1-nucleotidespacer). This motif has been shown to preferentially bind heterodimersof RXR and COUP-TF and heterodimers of RXR and the PPARs. DNA mobilityretardation experiments using various HRE sequences as competitordemonstrated that ARF6 preferentially recognizes DR-1 sites.

ARF6 has been identified as a heterodimeric complex of RXRα and PPARγ.It has been shown that PPARγ and RXRα form heterodimers on ARF6-bindingsites in vitro. Forced expression of these factors in transienttransfections is sufficient to activate the adipocyte-specific aP2enhancer in nonadipose cells such as fibroblasts. This activation ispotentiated by peroxisome proliferators, fatty acids, and 9-cis retinoicacid. Antiserum to RXRα specifically inhibits ARF6 activity in adipocytenuclear extracts.

Cotransfection of the RXRα expression vector and the PPARγ expressionvector has a synergistic effect to activate the aP2 enhancer innonadipose cells. Maximal activation of the aP2 enhancer is observedwhen both PPARγ, RXRα and their agonists are present.

Without being bound by any theory, applicant proposes that an RXRagonist affects glucose usage in tissues through the synergistic effectsof RXR and PPARγ heterodimers. The RXR/PPARγ heterodimers, whenactivated by an RXR agonist or a combination of an RXR agonist and aPPARγ agonist, induce adipogenesis and modulates the levels of glucoseand triglyceride uptake. Alternatively or in addition, the RXR/PPARγheterodimers, when activat by an RXR agonist or a combination of an RXRagonist and a PPARγ agonist, regulate signaling molecules secreted byadipose tissue such as tumor necrosis factor-α or leptin, which in turnmodulates glucose metabolism in other tissues.

Using RXR Agonists to Mimic or Enhance the Antidiabetic Effects ofThiazolidinediones

PPARα, β and γ all form heterodimers with RXRs. These RXR/PPARheterodimers bind to DNA and regulate transcription activity. RXRactivators cooperate with PPARα activators to activate the activity ofPPARα protein (Kliewer, et al. Nature 358:771-774 (1992) and Mukherjee,et al. Steroid Biochem. Molec. Biol. 51:157-166 (1994)).

In this invention, a similar synergistic activation was observed with aPPARγ activator and several RXR activators.

According to this invention, RXR agonists, e.g. LGD 1069, ALRT 1057 andLG 100268, may be utilized in the treatment of diabetes. We examinedfour independent parameters for the effects of RXR agonists, i.e.morphological changes, lipid accumulation, regulation of gene expressionand increased glucose uptake.

Two pre-adipocyte cell lines were used to test the theory of RXRactivation in the PPARγ/RXR heterodimer. 3T3-LI and C3H/10T1/2 cellswere obtained from ATCC and are derived from mouse embryo. They arecontact inhibited and can be induced to differentiate into adipocytecells containing large lipid droplets within the cytoplasm. Adipocytedifferentiation can be observe by oil red O staining which stains thelipid droplets within the cytoplasm red. The extent of adipocytedifferentiation can then be monitored by microscope observation.

For a more quantitative assay and rapid screening of compounds, a96-well plate assay was developed to quantitate the amount oftriglyceride produced by the differentiating adipocytes. In this assay,cells are grown as a monolayer to confluence on a 96-well plate andtreated with BRL 49653, insulin, and retinoids alone or in variouscombinations. These treatments induce differentiation to differentextent in both 3T3-LI and C3H/10T1/2 cells. The level of triglycerideaccumulation can then be measured via an enzymatic color reaction whichcan be read in a plate reader.

A third measure of adipocyte differentiation is to examine regulation ofgene expression. The mRNA expression levels of both PPARγ andlipoprotein lipase (LPL) have been shown to be modulated duringadipocyte differentiation. Northern blot analysis was used to dissectthe molecular aspects of how retinoids effect target genes ofdifferentiating adipocytes. PPARγ, lipoprotein lipase (LPL), and β-actin(loading control) mRNA levels were monitored after cells were treatedwith thiazolidinediones and retinoids.

A fourth indicator for the utility of a compound in treating NIDDM orinsulin-resistant diabetes is the compound's ability to enhanceinsulin-stimulated glucose uptake. Labeled 2-deoxyglucose (2-DOG, aglucose analog) assay was performed with a preadipocyte cell line in thepresence of insulin and a candidate compound to measure the level of2-DOG incorporation.

A) Retinoid Modulation of Lipid Accumulation in 3T3-LI Cells Stainedwith Oil Red O

Table 1 shows the percent of 3T3-LI cells that had differentiated intoadipocytes as observed by oil red O staining assay. BRL 49653 and LG100268 were used at 1 μM, insulin was used at 0.01 mg/mil. Wells treatedwith LG 100268 had bigger redder lipid droplets within the cytoplasm.

BRL 49653 and LG 100268 treatment alone induced 50% of the cells todifferentiate into adipocytes. This was increased dramatically with theaddition of insulin. Insulin in combination with BRL induced 80% of the3T3-LI cells into adipocytes while the combination of insulin and LG100268 induced 90% of the cells to differentiate. When BRL 49653 wasused in combination with LG 100268, an RXR agonist, the amount ofadipocytes differentiation was also increased dramatically. Other RXRagonists mimic the activity of LG 100268. For example, the addition ofALRT 1057 (pan agonist) or LGD 1069 (RXR specific agonist) incombination with BRL 49653 increased the amount of differentiation,albeit to a lesser extent than the strong RXR agonist LG 100268. Thecombination of insulin with BRL 49653 and LGD 1069 had a strongdifferentiating effect (95%) on the 3T3-LI cells.

B) Retinoid Modulation of Triglyceride Content in DifferentiatedAdipocytes

The retinoid modulation of lipid formation was quantitated by monitoringtriglyceride formation. FIGS. 1a and 1b show triglyceride accumulationin 3T3-LI cells treated with a retinoid (LG 100268 or LGD 1069) alone orin combination with a thiazolidinedione and insulin. Retinoids and BRL49653 were used at 1 μM, insulin was used at 0.01 mg/ml for allexperimental combinations.

Insulin, BRL 49653 and retinoids all induced some triglycerideaccumulation when used alone, with LG 100268 giving the largestresponse. The addition of retinoids (LGD 1069, LG 100268) with thethiazolidinedione (BRL 49653) to the assay increased the amount oftriglyceride accumulation in 3T3-LI differentiating adipocytes. This wasalso observed when BRL 49653 or LG 100268 was used in combination withinsulin. The largest accumulation of triglyceride was seen in the cellstreated with LG 100268, BRL 49653 and insulin together. Similar resultswere observed when LGD 1069 replaced LG 100268 in the study. Theseresults concur with those obtained in the oil red O staining assays.

C) PPARγ and LPL mRNA modulation in differentiating 3T3-LI cells

Adipocyte specific genes were monitored via northern blot analysis.FIGS. 2a and 2b show the expression pattern of LPL (lipoprotein lipase)mRNA and PPARγ mRNA in cells that were treated for 7 days with BRL 49653(1 μM), LG 100268 (1 μM) and insulin (0.01 mg/ml), alone or incombination.

Northern blot analysis shows an increase in the relative signalnormalized to β-actin of both LPL and PPARγ mRNA expression in cellstreated with either compound alone. There was a three to five foldincrease in mRNA levels for these adipocyte target genes demonstratingthat transcriptional regulation occurs with treatment by insulin, BRL49653 and LG 100268. Combination of insulin, BRL 49653 and LG 100268 didnot further enhance the mRNA level .

These data demonstrate that, in 3T3-LI cells, RXR agonists induceadipocyte differentiation by themselves or in combination withthiazolidinediones or insulin. RXR agonists enhance the activity ofthiazolidinediones and insulin. Three independent measurements supportthat RXR agonists contribute to the modulation of the RXR/PPARγheterodimer in regulating adipocyte differentiation and useful intreating NIDDM.

D) LG 100268 enhances insulin stimulated glucose uptake in 3T3-LI cells

A murine preadipocyte cell line, 3T3-L1, is widely used to study glucoseuptake, adipogenesis, and has been used in the characterization ofthiazolidinediones and other PPARγ activators. Insulin stimulated uptakeof labeled 2-deoxyglucose (2-DOG, a glucose analog) was observed in3T3-L1 cells treated with BRL 49653 or LG 100268.

3T3-L1 cells were treated with BRL 49653 (10 μM) or LG 100268 (1 μM) for10 days. Insulin was added to the cells at a concentration of 0.01 mg/mlfor 5 days, thereafter no insulin was added. Labeled 2-deoxyglucoseuptake assay was performed (Szalkowski, et al., J. Endocrin.136:1474-1481, 1995). The level of incorporated labeled 2-DOG wasnormalized to the amount of total cellular protein. About 5-foldincrease in 2-DOG uptake was observed in 3T3-L1 cells treated with BRL49653 alone. About 2-fold increase in 2-DOG uptake was observed in3T3-L1 cells treated with LG 100268 alone.

This experiment shows that an RXR agonist increases insulin mediatedglucose uptake in 3T3-L1 cells like a known insulin sensitizer, athiazolidinedione. It demonstrates directly that RXR agonists can beused to treat a major symptom of NIDDM, i.e. insulin resistance. OtherRXR agonists useful for the treatment of NIDDM can be confirmed usingthis assay.

E) Retinoid Modulation of Lipid Accumulation in C3H/10T1/2 Cells Stainedwith Oil Red O

To further assess the retinoid regulation of adipocyte function weexamined C3H/10T1/2 cells, which are mouse embryo fibroblast/multipotentstem cells that can be induced to differentiate into adipocytes ormuscle cells. Table 2 shows the percent of C3H/10T1/2 cells that havedifferentiated into adipocytes as observed by oil red O staining assay.Experiment A was conducted in the presence of BRL 49653. Experiment Bwas conducted in the presence of both BRL 49653 and insulin. ALRT 1057and LGD 1069 were used at 1 μM and TTNPB (RAR selective compound) wasused at 10 nM concentration. Insulin was used at 0.01 mg/ml; and BRL49653 was used at 0.1, 1 and 10 μM. C3H/10T1/2 cells were treated for 7days, cells were stained with oil red O and observed under themicroscope.

Morphological changes were observed when the cells were treated withretinoids alone, even though the cells did not fully differentiate intoadipocytes. BRL 49653 alone caused no more than 10 percent of the cellsto undergo adipocyte differentiation. However, when BRL 49653 was usedin combination with an RXR agonist, LGD 1069 or ALRT 1057, there was alarge increase in the amount of differentiation, the same effect wasseen when BRL 49653 was used in combination with insulin.

The RAR agonist, TTNPB (which does not activate RXR), did not have thesame effect as RXR agonist LGD 1069 and ALRT 1057. In fact, TTNPBinhibited the differentiation induced by BRL 49653 or BRL/insulin.

The above experiments show that a thiazolidinedione (BRL 49653) aloneinduces a minimal amount of differentiation in C3H/10T1/2 cells.However, when BRL 49653 is used in combination with a retinoid such asLG 100268, LGD 1069, or ALRT 1057, which are RXR agonists,differentiation is dramatically increased. This is not seen with thepure RAR agonist, TTNPB. These data support that the PPARγ/RXRheterodimers, which drive the adipocyte differentiation process, can beactivated and enhanced by the binding of an RXR agonist. RXR agonistsare useful for modulating the levels of glucose and triglyceride uptake.

Using RXR Agonists to Lower Levels of Glucose and Triglycerides inAnimal Models of NIDDM

(A) In vivo experiment with db/db mice

Animals: Strain Diabetic C57BLKS/J--m+/+db, 82 mice

Source: Jackson Lab

Stock number 000642

Genotype m+/+db xm+/+db

DOB Jun. 19, 1996 ±3 d, DOA Jul. 23, 1996 -34 d old

Date of study: Aug. 5, 1996-Aug. 21, 1996 44-63 d old

Mice were identified by an ear punch code (#1-82) and separated into 8groups (A-H). Each group consisted of 10 mice separated into 4 cageswith 2-3 mice/cage. Several mice were lost during the course of thestudy from fluid injections into the lungs resulting in 2 groups with 9mice/group. Control group C consisted of 12 mice. The mice were fedpelleted Purina Lab chow #5015 containing 3.83 kcal/g with a caloriccomposition of 56% carbohydrate, 26% fat and 18% protein. Food and waterwere provided ad libitum. Food intake/cage was measured over selectedperiods and expressed as g food consumed/100 g mouse/day.

On days of study, food was removed from the cages at selected intervalsof time between 6:15 and 7:00 AM. Animal weights were recorded 2 h afterstart of fast and blood samples taken after a 3 h fast. Blood was drawnfrom a cut at the tip of the tail and collected into a heparinizedcapillary tube (approx. 75 μl volume). After centrifugation, thehematocrit was read in a microcapillary reader, recorded and the tubebroken for recovery of plasma for analysis of glucose, triglyceride andinsulin concentration.

Blood samples were collected on days 0₋₁, 0, 3, 7, 10, and on the finalday of study, days 13-15. After collection of blood samples on day 0,animals were refed their Purina chow diet and subsequently gavaged withcontrol solution in group C and one of seven test solutions in groupsdesignated A, B, D-H. The volume administered was equivalent in eachgroup averaging 0.6 ml/42 g mouse (0.01429 ml/g). The various solutionswere gavaged daily to their respective groups based on the animalsweight taken that morning or the weight of the animal taken on thepreceding day of weighing. To assess alterations in plasma FFA, on day10 an addition 75 μl blood sample was collected into an EDTA-coatedcapillary tube immediately after collecting the basal heparinizedcapillary tube sample.

On the final day of study, mice were not gavaged with test solution. Thelast gavage was administered on the day preceding the final day, i.e.,day 12 for animals terminated on day 13, day 13 for animals terminatedon day 14 and day 14 for animals terminated on day 15. Terminal bloodwas collected by decapitation to provide serum for assessment of HDL.

Results:

FIG. 3 shows that BRL49653 and LG100268 each independently lowered thelevel of glucose in db/db mice. In addition, the combination of BRL49653and LG100268 lowered the glucose level more than each compound did byitself. BRL 49653 (1 mg/kg) and LG 100268 (20 mg/kg) lowered the levelof glucose by 40% by day 15 in comparison to control. Thethiazolidinedione BRL 49653 at 1 mg/kg showed similar efficacy. Thecombination of an RXR activator and a PPAR activator showed greaterefficacy, leading to almost 50% drop in the level of glucose. The effectof the combination was rapid, glucose levels were reduced by day 1 ofthe study and reached a steady state by day 4. This indicates a rapidresetting of the steady state levels of glucose homeostasis. Therefore,RXR activators enhance the efficacy of PPARγ activators, and vice vesa.

FIG. 4 shows that RXR activators lowered the level of triglycerides indb/db mice. LG 100268 (20 mg/kg) lowered the level of triglycerides 40%by day 15 of the study. BRL 49653 (1 mg/kg) showed similar efficacy. Thecombination of these two compounds worked even better.

FIG. 5 shows that RXR activators modulators increased the level of HDLcholesterol in db/db mice. LG 100268 (20 mg/kg) increasedHDL-cholesterol levels (20%) in comparison to controls in db/db mice.BRL 49653 caused an equivalent level of increase. The combination ofthese two compounds showed a higher increase. HDL-C was measured by theprecipitation method using kits obtained from Bohringer-Mannheim(catalog # 543004 and 427578).

(B) In vivo experiment with ob/ob mice

Animals: Strain Obese C57 BL/6J-Lep^(ob)(4), 121 mice

Source: Jackson Lab Stock number 000632

Genotype Lep^(ob) ⁴ /+x Lep^(ob) (4) /+

DOB May 22, 1996±3 d, DOA Jul. 2, 1996--41 d old

Date of study: Jul. 21, 1996-Aug. 2, 1996--49-70 d old

Mice were identified by an ear punch code (#1-121) and separated into 12groups. Each group consisting of 10 mice. (one mouse was terminatedbefore start of the study because of bad teeth causing initial weightloss. As in preceding studies, mice in each group were housed in 4 cages(2-3 mice/cage) and provided water and Purina Lab chow #505\15 adlibitum.

On days of study, food was removed from the cages at selected intervalsof time between 6:15 and 7:00 AM. Animal weights were recorded 2 h afterstart of fast and blood samples taken after a 3 h fast. Blood was drawnfrom a cut at the tip of the tail and collected into a heparinizedcapillary tube (approx. 75 μl volume). After centrifugation, thehematocrit was read in a microcapillary reader, recorded and the tubebroken for recovery of plasma for analysis of glucose, triglyceride andinsulin concentration.

Blood samples were collected on days 0₋₃, 0, 3, 6, 8, 10, 14±1 and finalcollections were made on days 15, 16 or 17. FFA samples were collectedon day 10 by drawing a second 75 μl blood sample in a non-heparinizedtube coated with EDTA. This tube was collected immediately after theheparinized tube collection.

Animals in groups H were administered control gavage solution (0.6 ml/42g) daily commencing on day 0 and ending on the day preceding the finalday. 11 test solutions were administered to animals in groups designatedA-G, I-L. All gavage solutions were administered after refeeding on daysin which mice were fasted for blood sampling.

Results:

FIG. 6 shows that RXR modulators lowered the level of triglycerides inob/ob mice. RXR activators LGD 1069 (30 mg/kg) and LG 100268 (20 mg/kg)lowered the level of triglycerides by 34% and 60% respectively in ob/obmice by day 14 of the study. BRL 49653 was also able to lower the levelof triglycerides, although not as efficacious as LG100268.

FIG. 7 shows that RXR modulators lowered the level of glucose in ob/obmice. RXR activators LGD 1069 at 30 mg/kg and LG 100268 at 20 mg/kglowered the level of glucose nearly 50% in comparison to control. Thelevel of glucose was reduced to almost euglycemic levels by day 14 ofthe study. BRL 49653 (0.4 mg/kg) showed similar efficacy.

FIG. 8 shows that RXR modulators, LGD 1069 (30 mg/kg) and LG 100268 (20mg/kg), lowered the level of insulin in ob/ob mice. LG100268 lowered thelevel of insulin by 66% by day 14 of the study. LGD 1069 showed lowerefficacy. There was a very rapid effect of the compounds since the levelof insulin started to drop in day 1.

Pharmaceutical Formulations and Modes of Administration

The particular compound that affects the disorders or conditions ofinterest can be administered to a patient either by themselves, or inpharmaceutical compositions where it is mixed with suitable carriers orexcipient(s). In treating a patient exhibiting a disorder of interest, atherapeutically effective amount of a agent or agents such as these isadministered. A therapeutically effective dose refers to that amount ofthe compound that results in amelioration of symptoms or a prolongationof survival in a patient.

The compounds also can be prepared as pharmaceutically acceptable salts.Examples of pharmaceutically acceptable salts include acid additionsalts such as those containing hydrochloride, sulfate, phosphate,sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate,cyclohexylsulfamate and quinate. (See e.g., PCT/US92/03736). Such saltscan be derived using acids such as hydrochloric acid, sulfuric acid,phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid,tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid,and quinic acid. These salts can be prepared by standard techniques. Forexample, the free base form of the compound is first dissolved in asuitable solvent such as an aqueous or aqueous-alcohol solution,containing the appropriate acid. The salt is then isolated byevaporating the solution. In another example, the salt is prepared byreacting the free base and acid in an organic solvent.

Carriers or excipients can be used to facilitate administration of thecompound, for example, to increase the solubility of the compound.Examples of carriers and excipients include calcium carbonate, calciumphosphate, various sugars or types of starch, cellulose derivatives,gelatin, vegetable oils, polyethylene glycols and physiologicallycompatible solvents.

In addition, the molecules tested can be used to determine thestructural features that enable them to act on the RXR/PPARγheterodimer, and thus to select molecules useful in this invention.Those skilled in the art will know how to design drugs from leadmolecules, using techniques such as those disclosed in PCT publicationWO 94/18959, incorporated by reference herein.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in human. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. Levels in plasma maybe measured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g. Fingl et al., in The Pharmacological Basis of Therapeutics, 1975,Ch. 1 p. 1). It should be noted that the attending physician would knowhow to and when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministrated dose in the management of the disorder of interest willvary with the severity of the condition to be treated and to the routeof administration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. Liposomes are spherical lipid bilayerswith aqueous interiors. All molecules present in an aqueous solution atthe time of liposome formation are incorporated into the aqueousinterior. The liposomal contents are both protected from the externalmicroenvironment and, because liposomes fuse with cell membranes, areefficiently delivered into the cell cytoplasm. Additionally, due totheir hydrophobicity, small organic molecules may be directlyadministered intracellularly.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used-pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions. The pharmaceuticalcompositions of the present invention may be manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levitating, emulsifying, encapsulating,entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Liposomes may be usedfor encapsulated delivery.

Pharmaceutical formulations disclosed or described in Boehm, et al.,WO94/15902 are incorporated by reference herein.

All publications referenced are incorporated by reference herein,including the nucleic acid sequences and amino acid sequences listed ineach publication. All the compounds disclosed and referred to in thepublications mentioned above are incorporated by reference herein,including those compounds disclosed and referred to in articles cited bythe publications mentioned above.

Other embodiments of this invention are disclosed in the followingclaims.

                  TABLE 1                                                         ______________________________________                                        Oil Red O Staining in 3T3-L1 Differentiating                                  Adipocytes                                                                                   Percent of Differentiated                                      Treatment      Cells                                                          ______________________________________                                        Control        0.1                                                            BRL 49653      50                                                             Insulin        20                                                             LG 100268      50                                                             BRL + Insulin  80                                                             BRL + LG 100268                                                                              90                                                             LG 100268 + Insulin                                                                          90                                                             ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Retinoid Modulation of Adipocyte differentiation in                           C3H/10T1/2 Cells                                                                            Experiment A                                                                              Experiment B                                                      (+BRL)      (+BRL + Insulin)                                                  % differentiated                                                                          % differentiated                                    Treatment     cells       cells                                               ______________________________________                                        BRL (10 μM)                                                                              10          80                                                  BRL (1 μM) 8           80                                                  BRL (0.1 μM)                                                                             5           60                                                  BRL (10 μM)                                                                              10          80                                                  +ALRT 1057(1 μM)                                                                         80          99                                                  +LGD 1069(1 μM)                                                                          80          99                                                  +TTNPB(10 nM) 5           50                                                  BRL (1 μM) 8           80                                                  +ALRT 1057(1 μM)                                                                         65          90                                                  +LGD 1069(1 μM)                                                                          65          90                                                  +TTNPB(10 nM) 2           30                                                  BRL (0.1 μM)                                                                             5           60                                                  +ALRT 1057(1 μM)                                                                         45          85                                                  +LGD 1069(1 μM)                                                                          45          90                                                  +TTNPB (10 nM)                                                                              0.2                                                             ______________________________________                                         Undifferentiated controls 0% differentiation.                            

What is claimed is:
 1. A method of treating hypertriglyceridemia in ahost suffering therefrom, comprising the step of administering to saidhost a pharmaceutically effective amount of an RXR agonist.
 2. A methodof increasing the level of HDL cholesterol in a host, comprising thestep of administering to said host a pharmaceutically effective amountof an RXR specific agonist.
 3. A method of preventinghypertriglyceridemia in a host, comprising the step of administering tosaid host a pharmaceutically effective amount of an RXR agonist.
 4. Amethod of treating cardiovascular disease in a host suffering therefrom,comprising the step of administering to said host a pharmaceuticallyeffective amount of an RXR specific agonist.
 5. A method of preventingcardiovascular disease in a host, comprising the step of administeringto said host a pharmaceutically effective amount of an RXR specificagonist.
 6. The method of claim 2, wherein said RXR agonist is an RXRspecific agonist.
 7. The method of claim 6, wherein said RXR specificagonist is2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-cyclopropyl]-pyridine-5-carboxylicacid or a pharmaceutically acceptable salt thereof.
 8. The method ofclaim 6, wherein said RXR specific agonist is4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-2-carbonyl]-benzoicacid or a pharmaceutically acceptable salt thereof.
 9. The method ofclaim 2, further comprising the step of administering to said host apharmaceutically effective amount of a PPARγ agonist.
 10. The method ofclaim 9, wherein said PPARγ agonist is a thiazolidinedione compound. 11.The method of claim 10, wherein said thiazolidinedione compound isselected from the group consisting of5-[[4-[2-(methyl-2-pyridinylamino)ethoxy]phenyl]methyl]-2,4-thiazolidinedione,troglitazone, pioglitazone, ciglitazone, WAY-120,744, englitazone, AD5075, darglitazone, and their pharmaceutically acceptable salts.
 12. Themethod of claim 2, further comprising the step of administering to saidhost a pharmaceutically effective amount of a compound selected from thegroup consisting of insulin, an insulin derivative, an insulinsecretagogue, an insulin sensitizer, and an insulin mimetic.
 13. Themethod of claim 9, further comprising the step of administering to saidhost a pharmaceutically effective amount of a compound selected from thegroup consisting of insulin, an insulin derivative, an insulinsecretagogue, an insulin sensitizer, and an insulin mimetic.
 14. Themethod of claim 2, wherein said RXR agonist is orally, topically,intravenously, transdermally, rectally, or parentally administered tosaid host.
 15. The method of claim 2, wherein said RXR agonist isadministered to said host in a pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient.
 16. The method ofclaim 9, wherein said RXR agonist and said PPARγ agonist areadministered to said host in a pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient.
 17. The method ofclaim 12, wherein said RXR agonist and said compound are administered tosaid host in a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or excipient.
 18. The method of claim 12, whereinsaid RXR agonist, said PPARγ agonist and said compound are administeredto said host in a pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient.
 19. The method ofclaim 15, 16, 17 or 18, wherein said pharmaceutical composition consistsof a single dosage unit.
 20. The method of claim 4, wherein said RXRagonist is an RXR specific agonist.
 21. The method of claim 20, whereinsaid RXR specific agonist is2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-cyclopropyl]-pyridine-5-carboxylicacid or a pharmaceutically acceptable salt thereof.
 22. The method ofclaim 20, wherein said RXR specific agonist is4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-2-carbonyl]-benzoicacid or a pharmaceutically acceptable salt thereof.
 23. The method ofclaim 4, further comprising the step of administering to said host apharmaceutically effective amount of a PPARγ agonist.
 24. The method ofclaim 23, wherein said PPARγ agonist is a thiazolidinedione compound.25. The method of claim 20, wherein said thiazolidinedione compound isselected from the group consisting of5-[[4-[2-(methyl-2-pyridinylamino)ethoxy]phenyl]methyl]-2,4-thiazolidinedione,troglitazone, pioglitazone, ciglitazone, WAY-120,744, englitazone, AD5075, darglitazone, and their pharmaceutically acceptable salts.
 26. Themethod of claim 4, further comprising the step of administering to saidhost a pharmaceutically effective amount of a compound selected from thegroup consisting of insulin, an insulin derivative, an insulinsecretagogue, an insulin sensitizer, and an insulin mimetic.
 27. Themethod of claim 23, further comprising the step of administering to saidhost a pharmaceutically effective amount of a compound selected from thegroup consisting of insulin, an insulin derivative, an insulinsecretagogue, an insulin sensitizer, and an insulin mimetic.
 28. Themethod of claim 4, wherein said RXR agonist is orally, topically,intravenously, transdermally, rectally, or parentally administered tosaid host.
 29. The method of claim 4, wherein said RXR agonist isadministered to said host in a pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient.
 30. The method ofclaim 23, wherein said RXR agonist and said PPARγ agonist areadministered to said host in a pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient.
 31. The method ofclaim 26, wherein said RXR agonist and said compound are administered tosaid host in a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or excipient.
 32. The method of claim 27, whereinsaid RXR agonist, said PPARγ agonist and said compound are administeredto said host in a pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient.
 33. The method ofclaim 29, 30, 31 or 32, wherein said pharmaceutical composition consistsof a single dosage unit.
 34. The method of claim 5, wherein said RXRagonist is an RXR specific agonist.
 35. The method of claim 34, whereinsaid RXR specific agonist is2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-cyclopropyl]-pyridine-5-carboxylicacid or a pharmaceutically acceptable salt thereof.
 36. The method ofclaim 34, wherein said RXR specific agonist is4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-2-carbonyl]-benzoicacid or a pharmaceutically acceptable salt thereof.
 37. The method ofclaim 5, further comprising the step of administering to said host apharmaceutically effective amount of a PPARγ agonist.
 38. The method ofclaim 5, wherein said PPARγ agonist is a thiazolidinedione compound. 39.The method of claim 38, wherein said thiazolidinedione compound isselected from the group consisting of5-[[4-[2-(methyl-2-pyridinylamino)ethoxy]phenyl]methyl]-2,4-thiazolidinedione,troglitazone, pioglitazone, ciglitazone, WAY-120,744, englitazone, AD5075, darglitazone, and their pharmaceutically acceptable salts.
 40. Themethod of claim 5, further comprising the step of administering to saidhost a pharmaceutically effective amount of a compound selected from thegroup consisting of insulin, an insulin derivative, an insulinsecretagogue, an insulin sensitizer, and an insulin mimetic.
 41. Themethod of claim 5, further comprising the step of administering to saidhost a pharmaceutically effective amount of a compound selected from thegroup consisting of insulin, an insulin derivative, an insulinsecretagogue, an insulin sensitizer, and an insulin mimetic.
 42. Themethod of claim 5, wherein said RXR agonist is orally, topically,intravenously, transdermally, rectally, or parentally administered tosaid host.
 43. The method of claim 5, wherein said RXR agonist isadministered to said host in a pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient.
 44. The method ofclaim 5, wherein said RXR agonist and said PPARγ agonist areadministered to said host in a pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient.
 45. The method ofclaim 40, wherein said RXR agonist and said compound are administered tosaid host in a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or excipient.
 46. The method of claim 41, whereinsaid RXR agonist, said PPARγ agonist and said compound are administeredto said host in a pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient.
 47. The method ofclaim 43, 44, 45 or 46, wherein said pharmaceutical composition consistsof a single dosage unit.